1. Field of the Invention
The invention pertains to methods and compositions for detecting anti-hepatitis E virus activity in a subject. The compositions include nucleic acids encoding mosaic polypeptides of hepatitis E virus and mosaic polypeptides of hepatitis E virus. The methods include serologic diagnosis of hepatitis E viral infection using the mosaic polypeptides of this invention.
2. Background Art
Hepatitis E virus (HEV) is a recently discovered agent of enterically transmitted non-A, non-B hepatitis (ET-NANB). The disease remains a serious problem in many developing countries. Unlike other agents of viral hepatitis, HEV infection is often associated with high mortality rates in infected pregnant women.
The first reported outbreak of ET-NANB hepatitis occurred in New Delhi, India in 1955. However, only after serologic tests for IgM anti-hepatitis A virus became available to exclude hepatitis A virus as the cause, was this very large outbreak recognized as ET-NANB hepatitis. Since that time epidemics of ET-NANB infection have been documented in many countries.
Until recently, the diagnosis of ET-NANB hepatitis outbreaks could only be based upon the absence of serologic markers of hepatitis A virus (HAV) and hepatitis B virus (HBV). Subsequently, specific tests for the detection of the ET-NANB hepatitis were based upon immune electron microscopy (IEM), in which a small volume of a stool suspension from acutely infected individuals is incubated with acute- or convalescent-phase sera and examined by electron microscopy (Bradley et al. PNAS USA 1987;84:6277-6281, 1987). IEM, thus identified 27-32 nm virus-like particles using acute and convalescent phase sera as the source of antibody. However, since most clinical specimens do not contain sufficient virus-like particles to visualize using IEM, this method is not useful for clinical or epidemiological analysis.
Three open reading frames (ORF) have been identified (Tam et al. Virology, 185:120-131, 1991). Two type-common HEV epitopes were identified at the C-terminus of proteins encoded by ORF2 and ORF3 (Reyes et al. Gastroenterologia Japonica 26 (suppl.3): 142-147, 1991b; Ichikawa et al. Immunol. 35:535-543, 1991). These epitopes were expressed as large hybrid proteins with beta-galactosidase or glutathione-S-transferase and were recognized in an enzyme immunoassay by antibodies from acute- and convalescent-phase sera obtained from experimentally infected cynomologus macaques (Reyes et al., in "Viral hepatitis C,D,E", T. Shikata, R. H. Purcell, T. Uchida (Eds.) Elsevier Science Publishers, NY, pp.237-245, 1991a) or humans (Goldsmith et al., Lancet 339:328-331, 1992). These hybrid proteins have the disadvantage that the chimeric part of protein can negatively influence folding, and thus, antibody recognition. Furthermore, individuals may have antibodies expressed to the chimeric sequences, resulting in false positive diagnoses.
Reyes et al (in "Viral hepatitis C,D,E", T Shikata, R. H. Purcell, T. Uchida (Eds.) Elsevier Science Publishers, NY, pp.237-245, 1991) demonstrated that a short fragment of the C-terminal region of the protein encoded by ORF3, obtained by expression of DNA derived from the HEV genome of the Burma strain did not react with sera from cynomologous macaques infected with the Mexico strain of HEV. Conversely, expressed recombinant protein derived from the Mexico strain did not react with sera from macaques infected with the Burma strain of HEV (Yarbough et al. J. Virol. 65:5790-5797, 1991). Sequence comparison of the two strains at the C-terminal region of ORF3 revealed a 78% homology (Yarbough et al., 1991). Thus, strain-specific immune responses of subjects can result in false negative diagnoses using the available technology.
A strategy for the construction of mosaic proteins retaining the antigenic reactivity of a natural prototype antigen has been recently applied to the hepatitis B surface antigen (HBsAg) (Kumar et al. Gene 110:137-144, 1992). This protein was composed of two antigenic epitopes from the preS-protein and a short region modeling the HBsAg conformational antigenic determinant "a". Antigenic properties of these epitopes can be successfully modeled with short synthetic peptides. The artificial HBV protein also retained the immunoreactivity of the introduced HBsAg antigenic epitopes.
Recently, an HCV antigen was constructed by joining 3 large segments of proteins (266, 363,and 119mers) into one polypeptide chain (Chien et al. Proc. Nat'l Acad. Sci. USA 89:10011-10015 (1992). There are also examples in the literature of successful expression of small antigenically active regions with carrier proteins. These antigens lack sensitivity.
None of the above-described HEV peptides or recombinant antigens provide a sensitive and specific means for diagnosing HEV infection. Thus, because of the lack of sensitivity and difficulty of performing the previously available tests, there exists a need for a rapid, simple and highly sensitive and specific diagnostic test for HEV infection.
The invention satisfies these needs with an artificial polypeptide containing a mosaic of antigenic epitopes from the ORF2 protein and from the ORF3 protein of the HEV Burmese and Mexican strains combined together. The "mosaic" protein is a valuable reagent for diagnostic tests for the detection of anti-HEV activity.